Biomolecular processes are extensively studied by employing fluorescent dyes that either bind non-covalently to a target in the system or, to gain specificity, are covalently linked to the investigated biomolecule. Changes in fluorescence intensity or wavelength indicate that a biochemical event has taken place. There is a large choice of fluorescent dyes whose signal is affected by several physico-chemical parameters, such as pH, hydrophobicity, oxidation state or ionic strength. To improve the strategy of using probes comprising a single label, quencher dyes have been developed to provide dual-labeled probes, in which the quencher is paired with the reporter dye to enhance the observable change in fluorescence. Typically, these probes have a closed (i.e. quenched) form in which the reporter and the quencher are close to each other in space and an open form (i.e. fluorescent) in which the reporter and the quencher are spatially separated.
The quencher can be a second fluorescent dye. In this case, the fluorescence of the reporter can be monitored alone, or both the increase in the fluorescence of the quencher and the decrease in fluorescence of the reporter are observed. An overlap between quencher and reporter fluorescence spectra may cause background noise, which necessitates dedicated care in the instrumental set-up and data analysis as well.
Dark quenchers (e.g. non-fluorescent dyes) offer a solution to this problem because they do not occupy an emission bandwidth. The dual-labeled probes including a reporter and a dark quencher are also called fluorogenic or turn-on probes, since a (bio-)chemical event causes their transition from a non-fluorescent to a (typically highly) fluorescent form.
Dabcyl (4-(4′-dimethylamino-phenylazo)benzoic acid) is a widely used dark quencher [1] in dual-labeled probes for a variety of biomolecular applications, like enzymatic catalysis and nucleic acid probes [2, 3].
Dabcyl is a molecule based on an azobenzene scaffold, which consists of two phenyl rings linked by an azo group (N═N) in which each nitrogen atom carries a non-bonding pair of electrons:

This aromatic system confers high hydrophobicity to dabcyl making it insoluble in aqueous solution. Therefore, stock solutions of dabcyl need to be prepared in DMSO.
The absorption band of dabcyl in the range of 400-550 nm overlaps with the emission band of many common fluorescent dyes such as EDANS (5-((2′-aminoethyl)amino)naphthalene-1-sulfonic acid; λem, Max=490 nm), monobromobimane (mBBr; λem, Max=480 nm), and many fluorescein, coumarin and rhodamine derivatives, e.g. carboxyfluorescein (FAM; λem=515 nm; in water), coumarine 1 (λem=448 nm; in ethanol), rhodamine 123 (λem=512 nm; in ethanol) to cite only a few.
Although dabcyl is one of the most popular acceptors for developing fluorescence resonance energy transfer-(FRET)-based biological probes, the very poor solubility in water set limits to its use in biological systems where the natural solvent is water. Although this hydrophobicity can be compensated by the hydrophilicity of the substrate to which dabcyl is linked (e.g. long DNA segments or peptide chains), it represents a real problem in case of comparatively small substrate (e.g. glutathione) in which this compensation is more difficult.
Solubility problems have been observed for several dabcyl-labeled substrates [4]. Incomplete dissolution leads to incorrect estimation of the concentrations and consequently wrong calculations of the stability and rate constants. Attempts to overcome the problems deriving from the insolubility have been done, e.g. by performing the enzymatic assays in mixture of water and DMSO [2].
Decreasing the hydrophobicity of a compound is usually done by adding either sulfonate or hydroxyl groups to the compound. The modification, however, must not lead to a change of the desired properties of the compound. In the case of dabcyl, it is imperative that the fluorescence properties, namely the function as dark quencher, are not changed. For example, the emitting properties of the compound must not be significantly affected by the modification. If the modification leads to a change of the emitting properties, the compound might become fluorescent itself and therefore is no longer suitable as a dark quencher. This effect is known to occur when hydroxyl groups are added to a compound. For example, the addition of one hydroxyl group transforms the weak fluorescence of phenylalanine in the red-shifted stronger fluorescence of tyrosine.
Furthermore, the modification must not lead to a significant change in the electrostatic profile of the compound, as its function is to interact with molecules in the context of biological systems, wherein the molecular interactions are often driven by electrostatics (e.g. enzymatic reactions). A change in the electrostatic profile is a known effect of the addition of sulfonate groups [5].
In addition, the modification must not lead to a significant structural change of the compound, which affects the interaction with molecules in the biological systems, where the compound is to be used. For example, it has to be prevented that catechols are formed by the modification, as the catechols strongly chelate metals (e.g. Fe(III)). The chelation leads to unwanted reactions that may interfere with the system under investigation. This aspect is particularly important for the investigation of enzymatic reactions in which metals are essential cofactors and for possible applications in vivo.
It was the problem to be solved by the present invention to provide a compound suitable as a dark quencher, which can be used in aqueous systems, is superior in spectroscopic properties compared to dark quenchers of the state of the art, and which is easier to handle.
This problem was solved by providing the compound 4-((4′-(dimethylamino)2′,6′-dihydroxyphenyl)azo)2-hydroxybenzoic acid, which is herein also called Hydrodabcyl. Hydrodabcyl is easier to handle compared to dabcyl as it is soluble in aqueous solutions. It is superior to dabcyl as it has superior quenching abilities due to a higher molar absorbance compared to dabcyl.
A further problem to be solved by the present invention was to provide an improved method for synthesizing Hydrodabcyl. This problem is solved by providing the method as described.